All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
The 65-kDa heat shock protein (HSP65) of the Mycobacterium Tuberculosis plays a significant role in the pathogenesis of autoimmune arthritis. Its effect is well exemplified in the experimental model of adjuvant arthritis (AA). AA can be induced in susceptible, inbred strains of rats such as Lewis or Wistar, by intracutaneous inoculation of heat killed mycobacteria suspended in Freunds adjuvant. AA can be passively transferred by a T-cell clone reactive to residues 180-188 of the HSP65 [Holoshitz, J. et al. Science 219:56-58 (1983)].
Evidence has been reported that protection from disease may be due to cellular responses to HSP65 [Lider, O. et al. Proc. Natl. Acad. Sci. 84:4577-4580 (1987); Moudgil, K. et al. J. Exp. Med. 185:1307-1316 (1997)], suggesting that this protein contains different epitopes which participate in both pathogenesis and acquisition of resistance. The inventors have previously shown that resistance to AA can also be conferred by antibodies against HSP65 and can be passively transferred by intravenous infusion of immunoglobulins from arthritis-resistant strains to arthritis-susceptible rats [Ulmansky, R., and Y. Naparstek, Eur. J. Immunol. 25:952-957 (1995)]. Further analysis defined the epitope specificity of the anti-HSP protective antibodies to amino-acid residues 31-46, designated as peptide-6 (also denoted by SEQ ID NO. 1) [Ulmansky, R. and Y. Naparstek, J. Immunol. 168: 6463-6469 (2002)]. Vaccination of Lewis rats with this peptide resulted in the production of antibodies against the whole molecule as well as resistance to disease induction.
The inventors have previously shown that polyclonal antibodies against peptide-6 stimulate IL-10 production by peripheral blood mononuclear cells (PBMCs) [Ulmansky (2002) ibid.]. The anti-inflammatory cytokine IL-10 plays an important role in innate immunity mostly due to its inhibitory effects, which allow suppression of inflammatory responses. Monoclonal, as well as chimeric and humanized anti-peptide-6 antibodies (Proximab) generated by the inventors were shown to retain this protective effect by binding to PBMCs and stimulating IL-10 secretion from the cells.
The inventors have now also shown that antibodies directed against peptide-6 interact not only with peptide-6, but moreover, they cross react directly with a surface ligand on monocytes, and this interaction is the key for comprehension of the mechanism of action of these antibodies. Following binding of the anti-peptide-6 antibodies to monocytes, there is activation of a signal transduction pathway that leads to an increase in production and secretion of cytokines, specifically IL-10 that as an anti-inflammatory cytokine, attenuates and inhibits inflammatory processes, thereby leading to amelioration and treatment of an inflammatory disorder. This tilts the balance between pro-inflammatory Th1 cytokines, such as tumor necrosis factor alpha (TNF-alpha), and anti-inflammatory Th2 cytokines, such as IL-10. Modulation of the Th1/Th2 balance towards a Th2 anti-inflammatory response by the anti-peptide-6 antibodies has been therefore shown by the present inventors as applicable in the treatment of inflammatory disorders.
As shown by the present invention, the cellular target of the anti-peptide-6 antibodies is the CAP1 protein. Cyclase-associated proteins (CAPs) are evolutionarily conserved proteins which function in regulation of the actin cytoskeleton and in signal transduction pathways. Mammals have two CAP genes, encoding the related CAP1 and CAP2. CAP1 shows a broad tissue distribution, whereas CAP2 is significantly expressed only in brain, heart and skeletal muscle, and skin. The cDNA of human CAP1 was identified, cloned and shown to encode a 475 amino acid protein that is homologous to the yeast CAP proteins.
The original CAP was isolated as a component of the Saccharomyces cerevisiae adenylyl cyclase complex that serves as an effector of Ras/cyclic AMP pathway during nutritional signaling. CAPs are multifunctional molecules that contain domains involved in several functions. The NH2 terminus is necessary and sufficient for cellular responsiveness to activated RAS proteins, while the COOH terminus is required for normal cellular morphology and growth control. Genetic studies in yeast have implicated CAPs in vesicle trafficking and endocytosis. CAPs play a developmental role in multi-cellular organisms, and studies of Drosophila have illuminated the importance of the actin cytoskeleton during eye development and in establishing oocyte polarity.
Human CAP1 is a component of actin-cofilin complex. Human CAP is a bifunctional protein with an N-terminal domain that binds to Ras-responsive adenylyl cyclase and a C-terminal domain that inhibits actin polymerization. CAP1 and its C-terminal domain were observed to facilitate filament elongation at the barbed end and to stimulate ADP-ATP exchange on globular (G) (monomeric) actin subunits, a process that regenerates easily polymerizable G-actin.
The direct interaction of the anti-peptide-6 antibodies with the CAP1 molecule that was first shown by the invention as leading to induction of IL-10 expression, probably through cAMP dependent protein kinase, reflect the involvement of CAP1 as a key element in this novel pathway. These findings establish the possibility of using the CAP1 as a target for immuno-modulation of this particular pathway. Moreover, anti-CAP1 antibodies were shown to induce IL-10 expression, demonstrating the feasibility of using a CAP1 binding compound as an immuno-modulator.
It is therefore an object of the invention to provide the use of a compound that specifically binds and interacts with CAP1, in methods and compositions for the treatment of immune-related disorders.
Another object of the invention is to provide the use of CAP1 and any fragments thereof, as an immuno-modulating compound.
In yet another object, the invention provides the use of CAP1 in a screening method for identification of immuno-modulating compounds. Such compounds may be useful in modulating the Th1/Th2 balance in a subject suffering of an immune-related disorder.
These and other objects of the invention will become apparent as the description proceeds.